Several methods for detecting enzyme activity, also called enzyme activity assays, are known in the art.
Examples of known enzyme activity assays are assays for detecting phytase activity and for protease activity. However, known phytase activity assay methods are based on the release of inorganic phosphate by phytase from inositol phosphate. The amount of phosphate released from the inositol phosphate by the phytase can be assayed by a number of known methods (for example see Fiske (1925) and Lowry (1946)). Kits for performing phytase activity assays based on the release of inorganic phosphate from inositol phosphate are commercially available (for example phytic acid (phytate) total phosphorous assay available from Megazyme International Ireland Limited).
There are several disadvantages of the known phytase activity assay methods. One disadvantage is that they are end point assay methods, meaning that the amount of inorganic phosphate can only be measured after the enzymic reaction has been stopped. This makes it difficult to do kinetic studies of phytase activity.
Another disadvantage of these known assays is that the assay background is complicated by inorganic phosphate that exists naturally in biological samples or is added thereto. As the level of inorganic phosphate released by phytase is measured to assess phytase activity, this variable background level of inorganic phosphate can make the results variable and unreliable, particularly when used on biological samples.
A third disadvantage of some prior art methods is the toxic nature of the reagents used, such as molybdate and vanadium.
Phytase activity can also be measured based on assaying the release of inositol from inositol phosphate when it is degraded by phytase. Inositol is assayed using inositol dehydrogenase by known methods (for example see Prestwich (1991)).
The disadvantage of this method is that an additional enzyme called phosphatase is needed to hydrolyze inositol monophosphate to inositol, as commercial phytases have little or no activity on inositol monophosphate. Assaying inositol can also be problematic because many biological samples have high background levels of inositol, which is one of the major polyols in many biological systems.
Phytase activity may also be assayed using calcium phytate as a substrate at pH 5.5 or above. Calcium phytate has a certain turbidity and hydrolysis of this substrate by phytase causes the turbidity to decrease. The substrate can be added to an agarose gel so that the phytase can be assayed using plate diffusion assay. The disadvantage of this method is that the turbidity of calcium phytate is low, and it has no turbidity at lower pH. In addition, calcium phytate is not an ideal substrate for commercial histidine acid phytases. Another disadvantage of the plate assay is that acid producing microbes will produce false results as the acid produced will dissolve the calcium phytate thereby forming clear halos (see Howson and Davis 1983).
A semi-quantitative antibody-based phytase assay is described in WO2007001895. This method uses antibodies that can bind to phytase to assay phytases. But a drawback of this method is that it can not reliably differentiate between active, partially-heated inactivated and heat-inactivated phytase, for example phytase that has been heat-inactivated during feed processing. The other disadvantages of this technology are that it is expensive to develop, it is time consuming, the results are unreliable, and can lead to false positive results. False positive results indicate either that there is phytase present when there is not, or that the phytase present is not active. This can lead to less phytase being added than is needed to give the required amount of phytase activity. If not enough phytase activity is present in the feed animals fed on the feed may not have enough available phosphate.
Feed or feed ingredients or feed mixtures (premix) may be tested to identify whether they contain phytase and how much phytase they contain. If there is not enough phytase in the feed or feed ingredient additional phytase can be added. False negative results in tests for phytase can lead to too much phytase being added to the feed, which is costly.
There are many known methods for the assay of protease activity. Many of them are based on synthetic peptides or peptide analogues often labelled with chromophores or fluorophores. The advantage of these methods is their high sensitivity and their capability to monitor protease activity kinetically.
A disadvantage of these methods is that information obtained with synthetic peptides can not be related to that obtained with natural proteins with a high degree of certainty as natural proteins often have complicated conformations which change with their environment.
Other methods use natural proteins as a substrate to detect protease activity. The disadvantage of using natural proteins as a substrate for proteases in the prior art methods is that monitoring of protein hydrolysis is usually done by indirect methods. This means that these methods are end point assays which measure the products of the reaction only after it has been stopped. These methods therefore do not lend themselves to doing kinetic studies on natural proteins.
An example of a protease activity assay that is done using a natural protein is a pepsin activity assay that can be performed using haemoglobin as a substrate. Pepsin activity can be related to the release of trichloroacetic acid (TCA) soluble aromatic amino acids and peptides. The method has to be an end point assay because the enzyme reaction has to be stopped using TCA before its products can be assayed.
The same is true for protease assays using casein, which is often cross-linked with chromophores. The reaction has to be stopped by raising the pH in order to separate soluble chromophores from insoluble substrate.
The present invention seeks to provide a useful assay method. In particular the present invention seeks to provide an assay method that is particularly useful in testing feed or feed ingredients or feed mixtures (premix) to identify whether they contain phytase and/or protease and, in some instances, the level thereof.